Worldwide enterotoxigenic E. coli (ETEC) cause an estimated 210 million episodes of diarrheal disease and 380,000 deaths annually. The impact of this pathogen is greatest among infants and children less than 5 years of age. Adherence of the pathogen to the intestinal mucosa of the host is essential for the establishment of an infection. Attachment is usually achieved by pili which bind specific host receptors. The expression of CS1 and CS2 pili is positively regulated at the level of transcription by Rns a member of the AraC/XylS family. There are two Rns binding sites immediately upstream of the CS1 pilin promoter, within the region expected for a typical prokaryotic activator. Rns also positively autoregulates its own expression, however it does so through a highly unusual arrangement of DNA binding sites. Rns requires two DNA binding sites for the activation of its own promoter Prns. One of these is site 1 which is centered 224.5 bp upstream of the transcription start site (TSS), considerably further upstream than expected. Even more unusual is the location of site 3, which is centered 83.5 bp downstream of the TSS. Only a few prokaryotic activators are known to have binding sites downstream of the -10 hexamer. However we have shown that the virulence regulators VirF from Shigella flexneri, CfaR from ETEC, and AggR from enteroaggregative E. coli are also capable of activating Prns and like Rns, each requires the downstream binding site to do so. To date Rns is the only virulence regulator within this group for which an in vitro system has been developed. Thus our in vitro system, coupled with complimentary genetic analysis, affords us an outstanding opportunity to use Rns as a model system for a group of related regulators. These regulators are essential for the virulence of several pathogenic species of bacteria that collectively kill over 2 million people, predominantly children and infants, every year. A detailed understanding of these homologous regulators may eventually lead to new applications for disease treatment and prevention, such as attenuation of bacterial virulence by targeting the function of conserved virulence regulators. Our proposed studies will also provide new and fundamentally important information about transcription and its regulation because of the unique features of Rns autoregulation. These include activation from a downstream binding site and the potential repression of Prns by nonproductive RNAP open (RPo) complexes. Studies in this proposal will determine the molecular mechanism of Rns positive autoregulation and the biological significance of Rns-independent, nonproductive RPo complexes near Prns.